This application supplements an earlier proposal designed to help understand the role which folate or pteridine coenzymes and substrates play in enzymatic systems. Structural factors commonly occurring in these coenzymes include a fully-reduced pyrazine ring and a substituent at the 5- position. In an enzymatic reaction, oxidation of the pterin usually occurs and, in certain cases, this is associated with substituent removal. A particular example is the conversion of deoxyuridylate to thymidylate by thymidylate synthetase. Affinity chromatographic procedures have been developed for the efficient isolation of this enzyme from an amethopterin-resistant strain of Lactobacillus casei. The column materials containing 5-fluoro-2'- deoxyuridine-5' -p-aminophenyphos-phate (1) coupled to aminohexyl-Sepharose will used to: (a) provide protein for structural and mechanistic studies; (b) isolate synthetases from various sources for gross structural comparative purposes; and (c) isolate synthetases from mammalian sources, where enzyme usually exists at only very low concentrations. A further objective is the synthesis of derivatives of the synthetase inhibitor (1) which contain fluorescent or photoaffinity labels which could aid in the structural characterization of the active-site of this enzyme.